The focus of this revised renewal application remains centered around the characterization of natural SIV reservoirs and associated human zoonotic risk. In the previous budget period, we developed non-invasive (fecal-based) SIV detection methods and used these to (i) identify the sooty mangabey reservoirs of epidemic HIV-2 (Santiago et al., J. Virol, 2005), (ii) trace the origin of pandemic and non-pandemic HIV-1 to distinct chimpanzee communities in southern Cameroon (Keele et al., Science, 2006), and (iii) document HIV-1 group O-like viruses in wild gorillas (Van Heuverswyn et al., Nature, 2006). In the current application, we propose to determine what additional reservoirs of human immunodeficiency viruses exist, what role if any the gorilla played as an intermediary in the HIV-1 group O zoonosis, and what adaptive changes occurred in SIVcpz following its transmissions to humans and gorillas. Specific Aims include: 1. To identify the ape reservoir of HIV-1 group O. Currently available SIVgor strains represent the closest known relatives of HIV-1 group O, but are too divergent to represent an immediate reservoir. To determine the primate source of HIV-1 group O, we will screen additional chimpanzee and gorilla populations in areas of known SIVcpz and SIVgor endemicity. We expect this expanded survey to uncover new SIVcpz and SIVgor strains, including viruses immediately proximate to HIV-1 group O. 2. To characterize the genetic diversity, evolutionary history and biological phenotype of SIVgor. The natural history of SIVgor infection in wild gorillas is completely unknown. In this aim, we will screen both western and eastern gorilla species for SIVgor infection, sequence a select number of complete SIVgor genomes, and generate replication competent SIVgor clones for biological analyses. These studies will elucidate the evolutionary history and biological properties of this newly discovered ape virus. 3. To determine whether chimpanzees and/or gorillas continue to serve as a reservoir for human infection. Using novel multiplex microbead immunoassays (MIA), we will screen existing banks of HIV ELISA and Western blot positive human sera from SIVcpz and SIVgor endemic areas for evidence of human infection by these ape viruses. This will address whether additional transmissions to humans have occurred but gone unrecognized, and better define SIVcpz and SIVgor lineages that are capable of infecting humans. 4. To identify viral determinants of cross-species transmission and human adaptation. In this aim, we will generate a panel of infectious molecular clones of SIVcpzPtt, SIVcpzPts and SIVgor, and compare their infectivity, envelope properties, growth potential, and cytopathicity in human and chimpanzee PBMC and macrophage cultures, human tonsil explant cultures, as well as in a recently developed novel humanized (BLT) mouse model. These studies are designed to identify primate lentiviral determinants associated with transmission and host adaptation.Project Narrative HIV/AIDS ranks as one of the most important infectious diseases to have emerged in recent history. This application will define the ape origins of HIV-1, and elucidate SIVcpz and SIVgor determinants associated with transmission and spread in humans. The documentation of ongoing zoonoses, along with the identification of the infecting virus strain, would have obvious public health implications and impact future AIDS treatment and vaccine trials.